An investigation of the mechanisms controlling expression of the methionine regulon of E. coli K12 is proposed. Specifically, four of the regulon genes, metB, metJ, metLM and metF, which are clustered between 87min and 88min on the genetic map of E. coli K12 are to be analyzed. The organization of these four genes into transcription units will be determined. To achieve this objective, a series of specialized lambda transducing phage which carry either all or portions of the four gene cluster have been isolated. Characterization of these phage will be continued through analysis of their biological activity within the methionine system as well as through restriction endonuclease analysis, DNA heteroduplex analysis and other electron microscopic studies of their chromosomes. As a probe of the transcriptional organization of the four met genes, RNA/DNA hybridization studies will be conducted using the separated strands of the met transducing phage and in vivo or in vitro synthesized mRNA. The technique of gene fusion will be utilized to identify nucleotide sequences involved in expression of the met gene cluster. Transposon mediated mutagenisis of specialized met transducing phage will be used to refine the physical map of the met genes as well as to search for polar effects on met gene expression. Met amber and deletion mutants will also be examined for polar effects. A search for promoter up type mutants of the metB gene will be conducted using poorly metabolized analogs of o-succinyl homoserine, the natural substrate of the gene product. One of the four genes, metJ, appears to code for an aporepressor protein which controls met regulon expression. A continuing study of the genetic properties of the metJ locus and of the sites of action of the metJ gene product is proposed. At appropriate stages in all these studies, in vivo results will be analyzed using a previously developed in vitro DNA directed protein synthesizing system which has been optimized for met gene expression. This system will also be used to search for the effector which activates the aporepressor.